Genetics is essentially an approach to understanding biological processes through the systematic elimination of gene function. Historically, the genetic approach has involved the development of xe2x80x9cscreensxe2x80x9d for mutations in genes which affect a specific phenotypic trait of an organism. The great advantage of this approach has been that no prior knowledge of the molecular nature of the genes involved is required because the xe2x80x9cscreenxe2x80x9d identifies the affected genes by marking them with mutations. The mutation involved is frequently a change in the gene""s sequence which results in a loss-of-function of the encoded gene product. Unfortunately the genetic approach has many limitations. Indeed the study of essential genes, required for cell viability, is exceedingly difficult using a purely genetic approach. As the famous molecular biologist David Botstein once put it, xe2x80x9cdeath is not a phenotype;xe2x80x9d an expression which encapsulates the frustration of attempting to study the function of essential genes. The implementation of xe2x80x9creverse geneticsxe2x80x9d in yeast (see e.g. Winston et al. (1983) Methods Enzymol 101: 211-28) and, later, in mammals (see e.g. Capecchi (1989) Science 244: 1288-92), has allowed the positive identification of a gene as essential through the inability to recover viable yeast haploid gene xe2x80x9cknockoutxe2x80x9d spores or homozygous recessive xe2x80x9cknockoutxe2x80x9d mice. Nevertheless, the exact biological processes in which the essential gene is involved are difficult to determine due to the inability to isolate and/or study the doomed knockout yeast spore or the inviable homozygous mouse zygote. Thus the downstream effects on specific aspects of cell function following removal of the essential gene product cannot be readily determined using these traditional xe2x80x9cknockoutxe2x80x9d studies. Furthermore, while traditional gene xe2x80x9cknockoutxe2x80x9d experiments may be useful in demonstrating that a given gene is essential for the life of the organism, they provide no data on precisely how important the gene is or to what extent so-called xe2x80x9csecond-site suppressingxe2x80x9d mutations can arise which restore cell viability following the removal of the essential gene. These considerations are important in the selection of targets for the rational design of, for example, antibiotic or chemotherapeutic pharmaceutical agents.
Others have endeavored to devise systems for the directed inactivation of a specific target gene in a host eucaryotic cell. For example, in an attempt to provide for a systematic means of deriving temperature-sensitive conditional alleles of a given gene target, Dohmen et al. have devised a temperature-sensitive xe2x80x9cdegronxe2x80x9d cassette that can be appended to any gene of interest and used to render it thermosensitive (Dohmen et al. (1994) Science 263: 1273-6). This approach could thus be applied in theory to any essential gene of interest. However, the generality with which the thermosensitive degron can be successfully applied to specific gene targets has yet to be determined and the necessity of relying upon thermal induction for the resulting system is a major drawback. Indeed, eucaryotic cells experience a transient heat-shock response which can have profound effects on some cellular processes such as transcription. Furthermore, the requirement for induction by heat shock precludes useful application to mammalian transgenic animal systems. Still other systems have been developed for the specific targeted removal of a host gene. Notably the Cre/lox system (see e.g. Sauer (1998) Mehods 14: 381-92) allows for the inducible deletion of a specific target gene through the action of the Cre site-specific DNA recombinase. Using this system, genetic switches can be designed to target ablation of a target gene in a specific tissue and at a specific time during development. One shortcoming of this method is that, following recombinational deletion of the targeted gene from the chromosome, the remaining mRNA and polypeptide products of the gene may only slowly be titrated out of the host cell through consecutive mitotic cell divisions and/or the eventual turnover of the mRNA and polypeptide by cellular ribonucleases and proteases. Thus it would be desirable to have a more rapid means for directly inactivating specific target genes in a host eucaryotic cell.
In general, the present invention provides a rapid and effective means for inactivating target genes, including target genes involved in important biological pathways. The invention also provides a system for the rapid and reliable repression of gene function regardless of whether the gene of interest is known or suspected of being an essential gene.
In one aspect, the present invention provides multiple means for the rapid and inducible elimination of gene function in a controlled and reproducible manner in a population of otherwise mitotically viable eucaryotic cell. The methods described include a method for rapidly repressing the transcription of a target gene through the action of an inducible repressor, a method for rapidly removing the polypeptide product of a target gene through directed proteolysis, and an integrated method in which both transcriptional repression and directed proteolysis occurs. In one embodiment, the method provides an inducible means for the passive removal of an mRNA product of a target gene (i.e. new target gene mRNA synthesis is blocked and the existing target gene mRNA is allowed to degrade through the natural turnover of the remaining target mRNA). In another embodiment, the method provides an inducible means for the active removal of a polypeptide product of a target gene (i.e. while new target gene polypeptide synthesis, or translation, is not blocked per se, the existing target gene polypeptide product is actively degraded by proteolysis). In yet another, preferred embodiment, the first and second embodiments are xe2x80x9cintegratedxe2x80x9d thereby allowing an optimal rate at which gene function can be eliminated by the simultaneous removal of both mRNA and polypeptide products of the gene.
The present invention thus provides a method of determining which genes represent effective targets for the design of antibiotic and/or chemotherapeutic agents. In particular, an array of essential genes can be screened to determine which are most vital to cell viability using the method of the invention. For example, essential genes which, when targeted for destruction by this two-pronged inducible repression system, result in the immediate death of the host cell, are likely to be effective targets for antibiotic or chemotherapeutic agents designed to stop cell growth. The present invention further provides a means of genetically modifying a population of cells so as to render them subject to killing by a normally benign inducing agent. This modification provides a convenient way to terminate or attenuate the physiological effects on a host organism of a population of bioengineered cells which have been delivered to the host. In this application, virtually any essential gene can be targeted for the inducible repressional shut-off of the present invention. In still other applications, a bioengineered cell population which produces a specific physiologically active gene product could be designed so that the gene product itself is subject to the inducible repressional shut-off system. When such a bioengineered cell population is introduced into a host, the delivery of the physiologically active gene product produced by the bioengineered cells can be adjusted throughout the lifetime of the host/cell combination by administration of a benign inducing agent.